THE MAILLARD REACTION BETWEEN RIBOSE 5-PHOSPHATE AND CELLULAR AMINES

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。糖和胺之间的美拉德反应是烘焙/油炸食品中产生的颜色、风味和香气的主要因素，也是生理系统中晚期糖基化终产物（AGEs）的产生的主要因素。核糖5-磷酸（R5P）是细胞中戊糖磷酸途径的一个组成部分，已被证明与简单胺发生美拉德褐变反应的速度比大多数其他常见糖和磷酸糖快约100倍。然而，目前对细胞胺和蛋白质的重要R5P糖化的潜力知之甚少。该项目的目标是全面表征R5P与胺反应的动力学和反应机理，特别关注反应对蛋白质交联和功能的影响。R5P （50 mM）在37℃时与简单胺/氨基酸和靶蛋白（溶菌酶、乳清蛋白、核糖核酸酶A、组蛋白、肌红蛋白）反应迅速，通常在数小时内发现初始相互作用，通常在一天内发生明显的褐变反应。天然凝胶和等电聚焦电泳模式表明碱性胺侧链（赖氨酸和精氨酸）被带负电荷的R5P基团快速取代。与简单的R5P/ n -乙酰赖氨酸系统相比，R5P/蛋白质系统的建模表明，与蛋白质的反应速率提高了。R5P最终被从溶液中去除，尽管其摩尔含量超过总蛋白胺水平的十倍以上，这表明R5P的重复降解反应伴随着初始胺组的随后再生。微滤样品的31P核磁共振（NMR）谱分析显示，随着时间的推移，R5P核中的磷酸盐会损失。蛋白质交联速度非常快，对于高碱性蛋白质（即组蛋白）的系统，在4小时内可以看到二聚体，对于更中性的蛋白质，在4天内可以看到二聚体。这项研究的意义加强了R5P可能是细胞条件下晚期糖基化终产物（AGEs）的重要来源的建议。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The Maillard reaction between sugars and amines is a principal contributor to the colors, flavors, and aromas generated in baked/fried foods and to the production of advanced glycation end-products (AGEs) in physiological systems. Ribose 5-phosphate (R5P), a component of the pentose phosphate pathway in cells, has been shown to undergo the Maillard browning reaction with simple amines at rates approximately 100 times faster than most other common sugars and sugar phosphates. The potential of significant R5P glycation of cellular amines and proteins is, however, currently poorly understood. The goal of the project was to fully characterize kinetics and reaction mechanisms of the reaction of R5P with amines with particular attention towards the effect of the reaction on protein cross-linking and function. R5P (50 mM) was shown to react rapidly with simple amines/amino acids and target proteins (lysozyme, lactalbumin, ribonuclease A, histones, myoglobin) at 37 C with initial interactions noted generally within hours and browning reactions obvious generally within a day. Native gel and isoelectric focusing electrophoretic patterns suggest a rapid replacement of basic amine side chains (lysines and arginines) with negatively charged R5P groups. Modeling of the R5P/protein system vs. a simple R5P/N-acetyl lysine system illustrates an enhanced rate for the reaction with proteins. R5P was eventually removed from solution despite a greater than ten-fold molar excess over total protein amine levels, thus indicating a repeated R5P-degrading reaction coupled with subsequent regeneration of the initial amine group. 31P nuclear magnetic resonance (NMR) spectroscopy analysis of microfiltrated samples of the reaction revealed phosphate loss from the R5P nucleus with time. Protein ¿ protein cross-linking was remarkably fast with dimers being seen within 4 h for systems with highly basic proteins (i.e. histones) and within four days for more neutral proteins. The implications of this investigation strengthen a proposal that R5P could be a significant source of advanced glycation end-products (AGEs) in cellular conditions.